Vectors for the treatment of friedreich&#39;s ataxia

ABSTRACT

The present invention provides gene therapies for the treatment of Friedreich&#39;s ataxia. Specifically, the present invention provides a nucleic acid, cloning vector and transfer vector for the production of an adeno-associated virus (AAV) vector. The nucleic acid comprises (i) a nucleic acid sequence encoding frataxin, (ii) a phospho-glycerate-kinase (PGK) promoter, and (iii) a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). The present invention also provides a pharmaceutical composition which comprises the AAV vector or nucleic acid. Also, the AAV vector, nucleic acid or pharmaceutical composition can be used as a medicament, specifically as a medicament for the treatment of Friedreich&#39;s ataxia.

TECHNICAL FIELD

The present invention can be included in the field of new therapeuticsfor the treatment of Freidreich's ataxia. Specifically the presentapplication relates to new products for gene therapy. The products arecapable of treating Friedreich's ataxia.

BACKGROUND ART

Friedreich's ataxia (FRDA; OMIM #229300) is a rare inheritedneurodegenerative disease that causes progressive damage to the nervoussystem resulting in symptoms ranging from gait disturbances and languageproblems to heart disease or cardiomyopathy. The disease was named afterthe physician Nikolaus Friedreich, who was the first to describe it inthe 1860s. Ataxia, referred to problems of motor coordination such asawkward movements and instability, occurs in Friedreich's ataxia bydegeneration of nervous tissue in the spinal cord and nerves thatcontrol the muscular movement of the arms and legs. The spinal cordshrinks with nerve cells losing part of their myelin sheath.Friedreich's ataxia, although uncommon, is the most common hereditaryataxia, ranging from 1 in 30,000-50,000 live births, with a prevalenceof 2-4 per 100,000. Both sexes are affected equally. Symptoms usuallybegin between 5 and 15 years of age but may, on rare occasions, appearas early as 18 months or as late as 50 years of age. The first symptomthat appears is usually difficulty walking, or gait ataxia. Ataxiagradually worsens and spreads slowly to the arms and then to the trunk.Foot deformities such as pes cavus, involuntary folding of the toes, orhammer toes may appear as early signs. Over time, the muscles begin toweaken and to be consumed, especially in the feet, legs and hands, andthe deformities develop. Other symptoms include loss of tendon reflexes,especially in the knees and ankles. There is often a gradual loss ofsensation in the extremities, which can spread to other parts of thebody. Dysarthria develops, and the patient gets tired easily. The rapid,rhythmic and involuntary movements of the eyes called nystagmus arecommon. Most people with Friedreich's ataxia develop scoliosis, which ifsevere, can affect breathing.

Other symptoms that can occur are chest pain, shortness of breath andpalpitations. These symptoms are the result of various forms of heartdisease that often accompany Friedreich's ataxia, such as hypertrophiccardiomyopathy, myocardial fibrosis, or heart failure. Heart rateabnormalities such as tachycardia and heart block are also common. About20% of people with Friedreich's ataxia develop carbohydrate intoleranceand 30% show diabetes mellitus. Some affected people lose hearing orsight. The speed of disease progression varies from person to person.Generally between 10 and 20 years after the onset of the first symptoms,the person is confined to a wheelchair, and in later stages of thedisease individuals become totally disabled. Life expectancy may beaffected. Many people with Friedreich's ataxia die in adulthood due toassociated heart disease, the most common cause of death. However, somepeople with less severe symptoms of Friedreich's ataxia sometimes liveto 60 or 70 years.

Neuroimaging such as MRI shows normal appearance in the early stages ofthe disease, although progressively reveals variable atrophy of thecervical spinal cord and cerebellum. This often shows atrophy of thesuperior peduncle. Electrophysiological studies show conductionvelocities greater than 40 m/s with absence or reduction of sensoryaction potentials, and absence of reflex H. At the neuropathologicallevel, marked atrophy of the spinal cord, posterior roots, occasionallyof the cerebellum, and cardiac hypertrophy are observed.Neurodegeneration is identified in peripheral sensory nerves withprogressive loss of large dorsal root ganglion sensory neurons, dorsalcolumn degeneration, transynaptic degeneration of neurons in Clarke'sspine and spinocerebellar fibers, pyramidal tracts, and atrophy of thegracile and cuneiform nuclei. Secondary lesions may include atrophy ofthe dentate nucleus in the cerebellum affecting large glutamatergicneurons, and atrophy of Betz cells and corticospinal tracts.

In 1996, the cause of Friedreich's ataxia was identified as a moleculardefect in the FXN gene located on chromosome 9 (9q21 band). The mutationconsists of an abnormal homozygous expansion of the GAA triplet locatedinside an ALU sequence in the first intron of the FXN gene. About 98% ofpatients with FRDA have 2 chromosomes with a number between 201 and1,700 GAA repeats (more frequently between 600 and 900) in each of them.However, 2-5% of patients with FRDA present a GAA expansion and a pointmutation in the FXN gene in composite heterozygosis (Table 1). To date,more than 17 different mutations in the FXN gene have been describedcapable of triggering FRDA. The GAA expansion in FRDA is very unstableduring meiosis and interferes with FXN gene transcription causingabnormally low levels of frataxin mRNA. GAA expansion would silencetranscription of the FXN gene and thus abolish frataxin expression byforming triple DNA structures or DNA-RNA hybrids, or both. Morerecently, it has been proposed that the GAA expansion would block thetransition from initiation to elongation of transcription due to theformation of heterochromatin-like structures in the vicinity of GAAhyperexpansion. It has also been proposed that GAA expansion would leadto epigenetic methylation of the CpG sites located in the 5′ upstreamregion of the FXN gene causing their silencing. Thus, as a consequenceof the GAA mutation in the FXN gene, a deficiency of the frataxinprotein occurs in FRDA.

TABLE 1 Most prevalent allelic variants identified in the FXN genecausing Friedreich′s ataxia. OMIM DISEASE ALLELIC VARIANT 606829.0001GAA expansion within intrón 1 606829.0002 Transversion within exon 3:LEU106X 606829.0003 Transition c.385-2A > G affecting splicing606829.0004 Missense variant: Ile154Phe in exon 4 606829.0005 Missensemutation: Gly130Val 606829.0006 Missense mutation affecting start codon:Met1Ile 606829.0007 Missense mutation: Trp173Gly 606829.0008 Deletion of1 nt in codon 75 provoking protein truncation 606829.0009 Deletion of 6nt and insertion of 15 nt (c.371_376del6ins15) in exon 3

The FXN gene encodes a small conserved mitochondrial protein thatcontains 210 amino acids (aa) in its precursor form, with a molecularweight of 23,135 Da named frataxin (Q16595). This precursor protein formcontains an N-terminal sequence of transit that directs it to themitochondrial matrix where the mitochondrial peptidase converts it intodifferent smaller isoforms (FXN42-210, FXN56-210, FXN81-210, FXN78-210)of the mature protein frataxin being the FXN81-210 of 130 aa and 14.2kDa the most abundant. In humans, frataxin is detected in themitochondrial matrix in association with its inner membrane in a largevariety of tissues, and the most abundant levels are identified incardiac tissue, spinal cord and dividing lymphoblasts, and remarkablythe lowest levels in the cerebellum. Frataxin has not been detected inthe cerebral cortex to date. Since the gene responsible for the diseasewas identified and with the generation of several animal models,different functions have been postulated for frataxin such asmitochondrial iron homeostasis, iron storage, response to oxidativestress, biogenesis of Fe—S clusters, modulation of mitochondrialaconitase activity and regulation of oxidative phosphorylation. In FRDA,frataxin deficiency produced by homozygosis expansion of the GAAexpansion in the FXN gene leads to insufficient biosynthesis of theiron-sulphur clusters necessary for electron transport in themitochondria and aconitase assembly leading to dysregulation ofmitochondrial function and mitochondrial iron accumulation byalterations of its homeostasis. Frataxin also modulates the DNA bindingcapacity of the protein aconitase 1 (ACO1), which in addition toparticipating in the cycle of citric acid in the mitochondrial matrix,regulates the uptake and utilization of cellular iron.

Several animal and cellular models for Friedreich's ataxia have beengenerated by genetic manipulation. Generating an AF model in the mousemimicking as close as possible the human disease has been an arduoustask, and currently there are 8 distinct murine models of FRDA.Unfortunately none of them present with the combination of all thephenotypic symptoms of FA in the same animal such as lesions in thedorsal root ganglia (DRG) and cerebellar dentate nuclei, although theyare useful for the study of isolated aspects of the molecularneurodegenerative process in FRDA and for the evaluation of differenttreatments in specific symptoms. The most used in pre-clinicaltreatments are the Prp-CreERT and YG8R mice. The Prp-CreERT micespecifically develop cerebellar and progressive sensory ataxia, the mostprominent neurological functions of Friedreich's Ataxia. Histologicalstudies in these animals show abnormalities of the spinal cord anddorsal root ganglia with absence of motor neuropathy, a hallmark ofhuman disease, as well as arborisation defects in Purkinje cells of thecerebellum. In contrast, YG8R transgenic mice in the absence of theendogenous murine frataxin protein exhibit slowly progressive FRDApathology. In contrast to humans, these mice do not show cardiacdeficits.

Among viral and non-viral vectors used in gene therapy for humanpathologies, vectors derived from adeno-associated virus (AAV) haveshown important clinical benefits and prolonged expressions in animalmodels of Gaucher disease, Fabry disease, Pompe disease, Metachromaticleukodystrophy, Niemann-Pick A disease and mucopolysaccharidosis I, II,III A, III B, IV and VII among others. Intravenous administration of AAVvectors in animal models of lysosomal storage diseases has led toincreases in enzyme activity of up to 16 times normal values in blood,liver, spleen, kidney and muscle, making them very useful for treatingthis type of pathologies. Over the past 10 years, AAVs have been thevectors of choice in most clinical trials conducted to treat central andperipheral nervous system pathologies(http://www.abedia.com/wiley/index.html). Importantly, no adverseeffects have been observed with these vectors to date and the resultsare very promising. Thus, several factors have made AAVs become theideal gene delivery vehicle for the central nervous system (CNS).Adeno-associated virus vectors comprising a frataxin sequence have beenused to treat FRDA in mice (Gérard et al., 2014. Mol Ther Methods ClinDev. 1: 14044; Chapdelaine et al., 2016. Gene Ther. 23(7): 606-614;Tremblay et al., 2015. Mol Ther. 23(Suppl. 1): pS153; WO 2016/150964).But, the AAV vectors disclosed in the prior art either over- orunder-express frataxin and do not reach all target cells and neuronsaffected in FRDA, which make them unsuitable for the efficient treatmentof FRDA and in particular its neurological signs. Thus, an AAV vectorexpressing the frataxin protein at a therapeutically effective amount isneeded.

At present there is no effective therapy for the treatment of FRDA andtherefore there is a need for new therapeutics for the treatment ofFRDA. It is an objective of the present invention to provide a suitabletherapy for treating FRDA.

FIGURES

FIG. 1. Evaluation of the SYN (synapsin) and FXN (frataxin; 1,255 bp)human promoters on frataxin (FXN) protein expression. To test the levelsof human frataxin (FXN) protein expression under the regulation of afragment of the endogenous promoter (phFXN), and the synapsin neuronalpromoter (phSYN), in comparison to the expression under thehigh-expressing constitutive promoter CMV, Human Neuroblastoma SH-SY5Ycells were transfected with empty vector (lane 1), or constructsencoding the human synapsin (phSYN) (lane 3), or a 1,255 bp fragment ofthe frataxin (phFXN) promoters (lane 4). Constructs 1.2 and 1.3 usingthe phSYN or the phFXN (1,255 bp) to express the human FXN codingsequence (hFXN CDS) did not result in expression of the recombinantfrataxin protein (rFXN). HA, hemagglutinin tag.

FIG. 2. Evaluation of the SYN (synapsin), NSE (neuron-specific enolase),and FXN (frataxin; 1,255 bp) human promoters together with the WPREsequence on frataxin (FXN) protein expression. The effect of a 320 bpfragment of the 5′end from FXN, and WPRE sequences on the expression ofFXN from the phSYN promoter previously shown in FIG. 1 (lane 3, 4 vs 8)and of phNSE (lane 6) was evaluated. The different constructs weretransfected into HEK cells and the lysates analysed 48 hrs aftertransfection. The efficiency of the WPRE sequences in the stabilizationof RNA is seen in the construct expressing FXN from the CMV promoter(lane 2). However, the addition and combination of the regulatoryelements shown above did not result in the expression of FXN from thephSYN, hpFXN, or phNSE. HA, hemagglutinin tag; iFXN, intermediate formFXN; mFXN, mature form FXN; WPRE, woodchuck hepatitis virusposttranscriptional regulatory element.

FIG. 3. Evaluation of the SYN (synapsin), NSE (neuron-specific enolase),and FXN (frataxin; 1,255 bp) human promoters together with the CMVenhancer and WPRE sequences on frataxin (FXN) protein expression. HEKand mouse neuroblastoma cells N2a were transfected with constructscomprising the CMV enhancer together with either SYN, NSE or FXN (1,255bp) promoters. The combination of the regulatory elements shown abovedid not result in the expression of FXN from the phSYN, phFXN, or phNSEpromoters. HA, hemagglutinin tag; iFXN, intermediate form FXN; mFXN,mature form FXN; WPRE, woodchuck hepatitis virus posttranscriptionalregulatory element.

FIG. 4. Evaluation of the effect of the human promoters SYN (synapsin),NSE (neuron-specific enolase), and FXN (frataxin; 1,255 bp) humanpromoters with addition of the KOZAK (5′) and WPRE (3′) sequences onfrataxin (FXN) protein expression. The different constructs weretransfected into HEK cells and the lysates analysed 48 hrs aftertransfection. The efficiency of the WPRE sequences in the stabilizationof RNA is seen in the construct expressing FXN from the CMV promoter(lane 1 vs 2). The further FXN expression enhancement by the addition ofthe kozak sequence and the 105 nt sequences between the promoter and theFXN coding sequence is seen in lanes 2 vs 3. However, the addition andcombination of the regulatory elements shown above did not result in theexpression of FXN from the phSYN, phFXN, or phNSE. HA, hemagglutinintag; iFXN, intermediate form FXN; mFXN, mature form; pFXN, precursorform; WPRE, woodchuck hepatitis virus posttranscriptional regulatoryelement.

FIG. 5. Evaluation of the combination of a defined linker between thehuman promoters PGK1 (phosphoglycerate kinase 1), NSE (neuron-specificenolase), SYN (synapsin) or FXN (frataxin; 1,255 bp) and the codingregion (CDS) of FXN in addition to the KOZAK (5′) and WPRE (3′)sequences on frataxin (FXN) protein expression. The different constructsshown were transfected into either N2a (A, B and E) or HEK (C and D)cells and the lysates were analysed 48 hrs after transfection.Expression of FXN was consistently detected from the phPGK, but not fromthe phNSE or phFXN (1,255 bp) promoter when using the 105 bp linkerbetween the promoter and the coding region (A, C, and D: Lane 4; B: lane5 and E: lane 8). HA, hemagglutinin tag; iFXN, intermediate form FXN;mFXN, mature form; pFXN, precursor form; WPRE, woodchuck hepatitis virusposttranscriptional regulatory element.

FIG. 6. Expression of luciferase (LUC) under the PGK1 (phosphoglyceratekinase 1) human promoter following intrathecal injection of the rAAV9vector (4.6×10¹² vg/Kg) in YG8R Tg/− and WT mice. The expression ofluciferase was detected after intraperitoneal injection of 150 μls ofD-luciferin at 150 mg/Kg mouse-weight 3.5 months after intrathecalinjection of the vector (A). Organs were dissected and luciferasesubstrate was added fresh before capturing the image (relative scalesshown after left and right panels (B)).

FIG. 7. In vivo expression of frataxin mRNA in 7 months-old miceintrathecally administered with rAAV9-PGK1-FXN. Frataxin mRNA levelswere quantified by qRT-PCR in liver, heart, lumbar dorsal root ganglia(DRG-L), lumbar spinal cord (SC-L), thorasic spinal cord (SC-T),cervical spinal cord (SC-C), cerebellum (Cb), and brain (frontal regionC1) tissues from 7-months-old YG8R hemizygous transgenic (Tg/−) orhomozygous (Tg/Tg) mice. YG8R hemizygous transgenic mice (Tg/−) wereadministered with either rAAV-Null (shown in black) or rAAV9-PGK1-FXN(shown in light grey) and the FRDA homozygous transgenic mice (Tg/Tg)(shown in dark grey) were not injected. Unlike the YG8R hemizygous mice(Tg/−) carrying two tandem copies of the human FXN gene with ˜82 and˜190 GAA trinucleotide sequence repeats in one of the chromosomes, thehomozygous YG8R mice contain the two tandem copies in each of thechromosomes. Neither the homozygous or the hemizygous YG8R mice for thehuman FXN express endogenous Fxn since they have a mouse Fxn knockoutgenetic background. The homozygous YG8R mice served as a higherexpressing control for the human FXN mRNA since it contains more copiesof the human transgene yet exhibiting normal functions. Mice wereinjected intrathecally at 2.5 months of age and human FXN-specificprimers were used to quantify FXN mRNA in the different tissues. Levelsof total FXN mRNA were normalized to levels of those detected in theYG8R hemizygous mice (Tg/−) treated with rAAV9-Null for each of thetissues analysed. Five months after intrathecal injection of therAAV9-PGK1-FXN vector, the levels of the FXN mRNA were higher (around1.3 and 3.2-fold) in the rAAV9-PGK1-FXN injected hemizygous YG8R mice(Tg/−) with the dosage tested but not higher than levels in thehomozygous YG8R mice (Tg/Tg) which in all tissues, compared with thelevels in Tg/− mice AAV-Null treated. A) Relative levels of human FXNmRNA in both females and males mice n=6 (3 females and 3 males). Folddifferences between Friedreich ataxia mice model YG8R mice injected withthe control AAV-null compared to the rAAV9-PGK1-FXN injected YG8R miceare statistically significant in liver, dorsal root ganglia and spinalcord (p=0.019, p=0.004, and p=0.024; denoted by asteriks *) withincreasing trends in all other tissues shown. More variability wasobserved in male compared to female mice with the dose used. B) Relativelevels of hFXN mRNA in female YG8R mice. Statistically significant folddifferences in frataxin levels are detected in all tissues studied asdenoted by asteriks and listed in the table below. The fold increases inthe rAAV9-PGK1-FXN injected mice were never higher than levels detectedin the Tg/Tg YG8R mice. Statistical significance, p<0.05*, p<0.005**

FIG. 8. In vivo expression of the recombinant frataxin protein from therAAV9-hPGK1-FXN vector after intrathecal injection. Lysates fromcultured cells N2a transfected with empty vector or the frataxinexpressing vector which was used as a positive control (FIGS. 8A and 8B:lanes 1 and 2). The endogenous FXN protein in the N2a cells and WT miceare detected with the anti-FXN antibody (A and B). The human FXN proteinfrom the transgene in the YG8R mice as well as the human recombinant FXNprotein in the injected mice are detected in both the liver and the micebrain (A and B). Incubation of the blots with the anti-FXN antibody forone hour begins to reveal the human FXN protein while overnight (o/n)incubation reveals both the mouse FXN in the WT mice, and the human FXNin the hemizygous (Tg/−) and homozygous mice (Tg/Tg). Levels ofexpression of the recombinant human FXN protein expressed from theinjected rAAV9-FXN are similar to the levels of the endogenous mouse FXNprotein in the WT mice. HA, hemagglutinin tag; iFXN, intermediate formFXN; mFXN, mature form FXN; WPRE, woodchuck hepatitis virusposttranscriptional regulatory element.

FIG. 9. Restoration of the clasping reflex in the YG8R mice over timefollowing injection with the rAAV9-FXN vector. Clasping of hindlimbs wasscored from 0 to 3 (see Methods), denoting less to more impairment, inhemizygous (Tg/−) mice and WT injected with rAAV9-FXN or rAAV9-nullvectors. Results for females and males together (n=10, 5 males and 5females for each group). Alterations in the clasping reflex are detectedas early as 4 months of age in the hemizygous YG8R mice. Followingintrathecal injection of the rAAV9-FXN vector the clasping reflexappears to normalize, indicating a restoration over time of thisneurological pathological phenotype. Asterisks (*) denote significantdifferences between values for WT and the Tg/− mice injected withrAAV9-null and the pound signs (#) denotes significant differencesbetween values obtained for the Tg/− mice injected with rAAV9-null andTg/− mice injected with rAAV9-FXN. *, #: P<0.05

FIG. 10. In vivo expression of recombinant frataxin protein in therAAV9-hPGK1-FXN injected mice restores the electrophysiologicalproperties at the different distances from the stimulus (D1-D4) of thecaudal nerve. Asterisks (*) denote significant differences between WTand Tg/− YG8R FRDA mice treated with rAAV9-null, while the pound (#)sign denotes significant differences between the rAAV9-null and therAAV9-FXN YG8R-treated mice. Intrathecal injection of the mice at 2.5months of age with the rAAV9-FXN vector prevents or slows the defects innerve conduction in the FRDA mouse model for FRDA (YG8R). WT, green(n=10); Tg/− YG8R FRDA mice treated with the rAAV9-null vector, grey(n=10); Tg/− YG8R FRDA mice treated with the rAAV9-FXN vector, blue(n=10). Measurements were obtained at 1 cm, 2 cm, 3 cm and 4 cm from thetail tip denoted as values for sites D1, D2, D3, and D4, respectively *, #: P<0.05.

FIG. 11. Preservation of dorsal root ganglia in the rAAV9-FXN treatedFRDA mouse model YG8R mice. The caliber of the dorsal root ganglialumbar appeared preserved as well as the mitochondria morphology in theFRDA mouse model treated with the rAAV9-FXN compared with the same micetreated with the rAAV9-null virus.

SUMMARY OF THE INVENTION

The present invention provides an adeno-associated virus (AAV) vectorcomprising a nucleic acid, wherein the nucleic acid comprises: (i) anucleic acid sequence encoding frataxin; (ii) a phospho-glycerate-kinase(PGK) promoter; and (iii) a woodchuck hepatitis virusposttranscriptional regulatory element (WPRE). The present inventionalso provides a nucleic acid comprising: (i) a nucleic acid sequenceencoding frataxin; (ii) a PGK promoter; and (iii) a WPRE; a cloningvector which comprises the nucleic acid and a transfer vector whichcomprises the nucleic acid. Further, the present invention encompassesthe use of the nucleic acid, cloning vector or transfer vector for theproduction of the AAV vector of the present invention. Also, the presentinvention provides a pharmaceutical composition and the use of the AAVvector, nucleic acid or pharmaceutical composition as a medicament,specifically as a medicament for the treatment of FRDA.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms “treatment” and “therapy”, as used in the present application,refer to a set of hygienic, pharmacological, surgical and/or physicalmeans used with the intent to cure and/or alleviate a disease and/orsymptoms with the goal of remediating the health problem. The terms“treatment” and “therapy” include preventive and curative methods, sinceboth are directed to the maintenance and/or reestablishment of thehealth of an individual or animal. Regardless of the origin of thesymptoms, disease and disability, the administration of a suitablemedicament to alleviate and/or cure a health problem should beinterpreted as a form of treatment or therapy within the context of thisapplication.

The term “therapeutically effective amount” refers to an amount ofmatter which has a therapeutic effect and which is able to treat FRDA.

The terms “individual”, “patient” or “subject” are used interchangeablyin the present application and are not meant to be limiting in any way.The “individual”, “patient” or “subject” can be of any age, sex andphysical condition.

As used herein, “pharmaceutically acceptable carrier” or“pharmaceutically acceptable diluent” means any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed and, without limiting the scope of the presentinvention, include: additional buffering agents; preservatives;co-solvents; antioxidants, including ascorbic acid and methionine;chelating agents such as EDTA; metal complexes (e.g., Zn-proteincomplexes); biodegradable polymers, such as polyesters; salt-formingcounterions, such as sodium, polyhydric sugar alcohols; amino acids,such as alanine, glycine, glutamine, asparagine, histidine, arginine,lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, andthreonine; organic sugars or sugar alcohols, such as lactitol,stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose,myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g.,inositol), polyethylene glycol; sulphur containing reducing agents, suchas urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol,[alpha]-monothioglycerol, and sodium thio sulphate; low molecular weightproteins, such as human serum albumin, bovine serum albumin, gelatin, orother immunoglobulins; and hydrophilic polymers, such aspolyvinylpyrrolidone.

The term “frataxin” refers to a protein which is encoded by the FXN geneand which is usually localized in the mitochondrion. Details of theprotein can be found in the UniProtKB database under accession numberQ16595.

The term “promoter” refers to a DNA sequence to which RNA polymerase canbind to in order to initiate transcription. The sequence may furthercontain binding sites for various proteins that regulate transcription,such as transcription factors. The promoter sequence may be composed ofdifferent promoter fragments (either different or the same fragments)that are localized closely in the DNA sequence and may be separated bylinkers or spacers. Such promoters are referred to as chimericpromoters. In a preferred embodiment, the term “promoter” refers to aphospho-glycerate-kinase (PGK) promoter.

The term “posttranscriptional regulatory element” refers to a DNAsequence that when transcribed creates a tertiary structure whichenhances or inhibits the expression of a protein.

The term “operably linked” refers to two or more nucleic acid sequencesthat are connected in a way that allows one nucleic acid sequence toinfluence another. For example, the PGK promoter and the WPRE areoperably linked to the nucleic acid sequence encoding frataxin so thatthe expression levels of frataxin are regulated by the PGK promoter andWPRE.

The term “functional variant” refers to nucleic or amino acids whosenucleic or amino acid sequence differs in one or more positions from theparental nucleic or amino acid sequence, whereby differences might beadditions, deletions and/or substitutions of nucleic acids or amino acidresidues, and which are still functional and therefore a suitable fortreating FRDA. A skilled person may determine functional variants byseeking homologues with a BLAST search or by studying the variability ofthe protein or gene in a population.

The term “Adeno-associated virus” refers to a small virus which infectshumans and some other primate species. A “vector” is any vehicle whichcan be used to artificially carry foreign genetic material into a cell.Thus, an “AAV vector” refers to a recombinant AAV which carries anucleic acid into a cell.

AAV Vector

In a first aspect, the present invention provides an adeno-associatedvirus (AAV) vector comprising a nucleic acid, wherein the nucleic acidcomprises: (i) a nucleic acid sequence encoding frataxin; (ii) aphospho-glycerate-kinase (PGK) promoter; and (iii) a woodchuck hepatitisvirus posttranscriptional regulatory element (WPRE); wherein (ii) and(iii) are operably linked to and regulate the expression of (i).

SEQ ID NO: 1 refers to the following sequence:

GAATTCCGGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTCTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCAGGGCGCGCCGAGAGCAGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAATC ACCGACCTCTCTCCCCAG

In a preferred embodiment, the PGK promoter comprises SEQ ID NO: 1 or asequence which is at least 75% identical to SEQ ID NO: 1. Preferably thePGK promoter comprises SEQ ID NO: 1 or a sequence which is at least 75%,80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98% or 99% identical to SEQ IDNO: 1. More preferably, the PGK promoter is SEQ ID NO: 1 or a sequencewhich is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98% or99% identical to SEQ ID NO: 1.

The sequence identity between two sequences can be determined throughconventional methods. For example, by using standard alignmentlogarithms known in the state of the art such as BLAST (Altschul et al.,1990. J Mol Biol. 215(3): 403-10). In a preferred embodiment, thesequence identity between two sequences is determined using BLAST.

SEQ ID NO: 2 refers to the following sequence:

TCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG

In a preferred embodiment, the WPRE comprises SEQ ID NO: 2 or a sequencewhich is at least 75% identical to SEQ ID NO: 2. Preferably WPREcomprises SEQ ID NO: 2 or a sequence which is at least 75%, 80%, 85%,89%, 90%, 91%, 92%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 2. Morepreferably, the WPRE is SEQ ID NO: 2 or a sequence which is at least75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98% or 99% identical to SEQID NO: 2.

SEQ ID NO: 3 refers to the following sequence:

ATGTGGACTCTCGGGCGCCGCGCAGTAGCCGGCCTCCTGGCGTCACCCAGCCCGGCCCAGGCCCAGACCCTCACCCGGGTCCCGCGGCCGGCAGAGTTGGCCCCACTCTGCGGCCGCCGTGGCCTGCGCACCGACATCGATGCGACCTGCACGCCCCGCCGCGCAAGTTCGAACCAGAGAGGTCTCAACCAGATTTGGAATGTCAAAAAGCAGAGTGTCTATTTGATGAATTTGAGGAAATCTGGAACTTTGGGCCACCCAGGCTCTCTAGATGAGACCACCTATGAAAGACTAGCAGAGGAAACGCTGGACTCTTTAGCAGAGTTTTTTGAAGACCTTGCAGACAAGCCATACACGTTTGAGGACTATGATGTCTCCTTTGGGAGTGGTGTCTTAACTGTCAAACTGGGTGGAGATCTAGGAACCTATGTGATCAACAAGCAGACGCCAAACAAGCAAATCTGGCTATCTTCTCCATCCAGTGGACCTAAGCGTTATGACTGGACTGGGAAAAACTGGGTGTACTCCCACGACGGCGTGTCCCTCCATGAGCTGCTGGCCGCAGAGCTCACTAAAGCCTTAAAAACCAAACTGGACTTGTCTTCCTTGGCCTATTCCGGAAAAGATGCTT

In a preferred embodiment, the nucleic acid sequence encoding frataxincomprises SEQ ID NO: 3 or a sequence which is at least 75% identical toSEQ ID NO: 3 and is a functional variant of frataxin. Preferably, thenucleic acid sequence encoding frataxin comprises SEQ ID NO: 3 or asequence which is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%,98% or 99% identical to SEQ ID NO: 3. More preferably, the nucleic acidsequence encoding frataxin is SEQ ID NO: 3 or a sequence which is atleast 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98% or 99% identicalto SEQ ID NO: 3.

In a preferred embodiment, the nucleic acid further comprises: a linkerbetween the promoter and the nucleic acid sequence encoding frataxin,wherein the linker consists of or comprises SEQ ID NO: 6 or a sequencewhich is at least 75% identical to SEQ ID NO: 6. Preferably, the linkerconsists of or comprises SEQ ID NO: 6 or a sequence which is at least75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98% or 99% identical to SEQID NO: 6.

SEQ ID NO: 6 refers to the following sequence:

TGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGCCG CCACC

In a preferred embodiment, the nucleic acid further comprises a kozaksequence and the kozak sequence is also operably linked to and regulatesthe expression of the nucleic acid sequence encoding frataxin. A kozaksequence is a sequence which occurs in eukaryotic mRNA and has theconsensus sequence gccRccAUGG, wherein R is a purine, lower-case lettersdenote the most common base at a position where the base cannevertheless vary and upper-case letters are highly conserved.

SEQ ID NO: 4 refers to the following sequence:

GAATTCCGGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTCTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCAGGGCGCGCCGAGAGCAGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAATCACCGACCTCTCTCCCCAGTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGCCGCCACCATGTGGACTCTCGGGCGCCGCGCAGTAGCCGGCCTCCTGGCGTCACCCAGCCCGGCCCAGGCCCAGACCCTCACCCGGGTCCCGCGGCCGGCAGAGTTGGCCCCACTCTGCGGCCGCCGTGGCCTGCGCACCGACATCGATGCGACCTGCACGCCCCGCCGCGCAAGTTCGAACCAGAGAGGTCTCAACCAGATTTGGAATGTCAAAAAGCAGAGTGTCTATTTGATGAATTTGAGGAAATCTGGAACTTTGGGCCACCCAGGCTCTCTAGATGAGACCACCTATGAAAGACTAGCAGAGGAAACGCTGGACTCTTTAGCAGAGTTTTTTGAAGACCTTGCAGACAAGCCATACACGTTTGAGGACTATGATGTCTCCTTTGGGAGTGGTGTCTTAACTGTCAAACTGGGTGGAGATCTAGGAACCTATGTGATCAACAAGCAGACGCCAAACAAGCAAATCTGGCTATCTTCTCCATCCAGTGGACCTAAGCGTTATGACTGGACTGGGAAAAACTGGGTGTACTCCCACGACGGCGTGTCCCTCCATGAGCTGCTGGCCGCAGAGCTCACTAAAGCCTTAAAAACCAAACTGGACTTGTCTTCCTTGGCCTATTCCGGAAAAGATGCTTTGCCCACCTAGGGATCGGATCCCCGGGTACCGAGCTCGAATTCTGCAGATATCCAGCACACTTTGCCTTTCTCTCCACAGGTGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG

In a preferred embodiment, the sequence of the nucleic acid whichcomprises (i), (ii) and (iii) is SEQ ID NO: 4 or a sequence which is atleast 75% identical to SEQ ID NO: 4. Preferably, the nucleic acid whichcomprises (i), (ii) and (iii) is SEQ ID NO: 4 or a sequence which is atleast 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98% or 99% identicalto SEQ ID NO: 4.

In a preferred embodiment, the nucleic acid further comprises a PolyAsignal. Preferably, the PolyA signal is at least 50, 100, 150 or 228 bplong. More preferably the PolyA signal is at least 228 bp long. Mostpreferably, the PolyA signal is 228 bp long.

In a preferred embodiment, the nucleic acid further comprises one ormore inverted terminal repeat (ITR) sequences. Preferably the nucleicacid comprises two ITR sequences. More preferably, the ITR sequencesflank the rest of the components of the nucleic acid.

SEQ ID NO: 5 refers to the following sequence:

CTGGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGAATTCCGGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTCTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCAGGGCGCGCCGAGAGCAGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAATCACCGACCTCTCTCCCCAGTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGCCGCCACCATGTGGACTCTCGGGCGCCGCGCAGTAGCCGGCCTCCTGGCGTCACCCAGCCCGGCCCAGGCCCAGACCCTCACCCGGGTCCCGCGGCCGGCAGAGTTGGCCCCACTCTGCGGCCGCCGTGGCCTGCGCACCGACATCGATGCGACCTGCACGCCCCGCCGCGCAAGTTCGAACCAGAGAGGTCTCAACCAGATTTGGAATGTCAAAAAGCAGAGTGTCTATTTGATGAATTTGAGGAAATCTGGAACTTTGGGCCACCCAGGCTCTCTAGATGAGACCACCTATGAAAGACTAGCAGAGGAAACGCTGGACTCTTTAGCAGAGTTTTTTGAAGACCTTGCAGACAAGCCATACACGTTTGAGGACTATGATGTCTCCTTTGGGAGTGGTGTCTTAACTGTCAAACTGGGTGGAGATCTAGGAACCTATGTGATCAACAAGCAGACGCCAAACAAGCAAATCTGGCTATCTTCTCCATCCAGTGGACCTAAGCGTTATGACTGGACTGGGAAAAACTGGGTGTACTCCCACGACGGCGTGTCCCTCCATGAGCTGCTGGCCGCAGAGCTCACTAAAGCCTTAAAAACCAAACTGGACTTGTCTTCCTTGGCCTATTCCGGAAAAGATGCTTTGCCCACCTAGGGATCGGATCCCCGGGTACCGAGCTCGAATTCTGCAGATATCCAGCACACTTTGCCTTTCTCTCCACAGGTGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGAATTCGAGCTCGGTACGATCAGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTGGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAG GGGTTCCT

In a preferred embodiment, the nucleic acid is SEQ ID NO: 5 or asequence which is at least 75% identical to SEQ ID NO: 5. Preferably,the nucleic acid is SEQ ID NO: 5 or a sequence which is at least 75%,80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98% or 99% identical to SEQ IDNO: 5.

In a preferred embodiment, the vector allows for the expression of atherapeutically effective amount of frataxin in a patient who suffersfrom Friedreich's ataxia. Thus, the vector delivers the nucleic acid toa patient's cells where the nucleic acid then expresses frataxin at atherapeutically effective amount.

In a preferred embodiment, the AAV vector is an AAV serotype 9 vector,i.e. an AAV-9 vector.

Nucleic Acid

In a second aspect, the present invention provides a nucleic acidcomprising: (i) a nucleic acid sequence encoding frataxin; (ii) a PGKpromoter; and (iii) a WPRE; wherein (ii) and (iii) are operably linkedto and regulate the expression of (i).

In a preferred embodiment, the PGK promoter comprises SEQ ID NO: 1 or asequence which is at least 75% identical to SEQ ID NO: 1. Preferably thePGK promoter comprises SEQ ID NO: 1 or a sequence which is at least 75%,80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98% or 99% identical to SEQ IDNO: 1. More preferably, the PGK promoter is SEQ ID NO: 1 or a sequencewhich is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98% or99% identical to SEQ ID NO: 1.

In a preferred embodiment, the WPRE comprises SEQ ID NO: 2 or a sequencewhich is at least 75% identical to SEQ ID NO: 2. Preferably WPREcomprises SEQ ID NO: 2 or a sequence which is at least 75%, 80%, 85%,89%, 90%, 91%, 92%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 2. Morepreferably, the WPRE is SEQ ID NO: 2 or a sequence which is at least75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98% or 99% identical to SEQID NO: 2.

In a preferred embodiment, the nucleic acid sequence encoding frataxincomprises SEQ ID NO: 3 or a sequence which is at least 75% identical toSEQ ID NO: 3 and is a functional variant of frataxin. Preferably, thenucleic acid sequence encoding frataxin comprises SEQ ID NO: 3 or asequence which is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%,98% or 99% identical to SEQ ID NO: 3. More preferably, the nucleic acidsequence encoding frataxin is SEQ ID NO: 3 or a sequence which is atleast 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98% or 99% identicalto SEQ ID NO: 3.

In a preferred embodiment, the nucleic acid further comprises: a linkerbetween the promoter and the nucleic acid sequence encoding frataxin,wherein the linker consists of or comprises SEQ ID NO: 6 or a sequencewhich is at least 75% identical to SEQ ID NO: 6. Preferably, the linkerconsists of or comprises SEQ ID NO: 6 or a sequence which is at least75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98% or 99% identical to SEQID NO: 6.

In a preferred embodiment, the nucleic acid further comprises a kozaksequence and the kozak sequence is also operably linked to and regulatesthe expression of the nucleic acid sequence encoding frataxin.

In a preferred embodiment, the sequence of the nucleic acid whichcomprises (i), (ii) and (iii) is SEQ ID NO: 4 or a sequence which is atleast 75% identical to SEQ ID NO: 4. Preferably, the nucleic acid whichcomprises (i), (ii) and (iii) is SEQ ID NO: 4 or a sequence which is atleast 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98% or 99% identicalto SEQ ID NO: 4.

In a preferred embodiment, the nucleic acid further comprises a PolyAsignal. Preferably, the PolyA signal is at least 50, 100, 150 or 228 bplong. More preferably the PolyA signal is at least 228 bp long. Mostpreferably, the PolyA signal is 228 bp long.

In a preferred embodiment, the nucleic acid further comprises one ormore inverted terminal repeat (ITR) sequences. Preferably the nucleicacid comprises two ITR sequences. More preferably, the ITR sequencesflank the rest of the components of the nucleic acid.

In a preferred embodiment, the nucleic acid is SEQ ID NO: 5 or asequence which is at least 75% identical to SEQ ID NO: 5. Preferably,the nucleic acid is SEQ ID NO: 5 or a sequence which is at least 75%,80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98% or 99% identical to SEQ IDNO: 5.

Cloning Vector

In a third aspect, the present invention provides a cloning vector whichcomprises the nucleic acid according to any one of the previouslydescribed embodiments and additional nucleic acid elements for promotingreplication of the cloning vector in a bacterial cell.

The term “cloning vector” refers to any vector that is suitable forcloning, which generally involves the presence of restriction sites, anorigin of replication for bacterial propagation and a selectable marker.

The cloning vector of the invention comprises the nucleic acid of theinvention and can preferably be used to produce the transfer vector orAAV vector of the invention.

Transfer Vector

In a fourth aspect, the present invention provides a transfer vectorwhich comprises the nucleic acid according to any one of the previouslydescribed embodiments and additional nucleic acid elements for promotingintegration or transposition of the transfer vector into an AAV vector,preferably an AAV-9 vector.

The term “transfer vector” refers to a vector that is suitable forintegration or transposition in an AAV vector. The transfer vector thusgenerally permits the insertion of genetic information into an AAVvector.

The transfer vector of the invention comprises the nucleic acid of theinvention and can preferably be used to produce the AAV vector of theinvention.

Use of the Nucleic Acid, Cloning Vector and Transfer Vector

In a fifth aspect, the present invention provides the use of the nucleicacid of the present invention, the cloning vector of the presentinvention or the transfer vector of the present invention for theproduction of an AAV vector according to any one of the embodimentspreviously described.

Pharmaceutical Composition

In a sixth aspect, the present invention provides a pharmaceuticalcomposition comprising the AAV vector of the present invention or thenucleic acid of the present invention and a pharmaceutically acceptablecarrier or diluent.

A pharmaceutical composition as described herein may also contain othersubstances. These substances include, but are not limited to,cryoprotectants, lyoprotectants, surfactants, bulking agents,anti-oxidants, and stabilizing agents. In some embodiments, thepharmaceutical composition may be lyophilized.

The term “cryoprotectant” as used herein, includes agents which providestability to the AAV vector against freezing-induced stresses, by beingpreferentially excluded from the AAV vector's surface. Cryoprotectantsmay also offer protection during primary and secondary drying andlong-term product storage. Non-limiting examples of cryoprotectantsinclude sugars, such as sucrose, glucose, trehalose, mannitol, mannose,and lactose; polymers, such as dextran, hydroxyethyl starch andpolyethylene glycol; surfactants, such as polysorbates (e.g., PS-20 orPS-80); and amino acids, such as glycine, arginine, leucine, and serine.A cryoprotectant exhibiting low toxicity in biological systems isgenerally used.

In one embodiment, a lyoprotectant is added to a pharmaceuticalcomposition described herein. The term “lyoprotectant” as used herein,includes agents that provide stability to the AAV vector during thefreeze-drying or dehydration process (primary and secondaryfreeze-drying cycles), by providing an amorphous glassy matrix and bybinding with the AAV vector's surface through hydrogen bonding,replacing the water molecules that are removed during the dryingprocess. This helps to minimize product degradation during thelyophilization cycle, and improve the long-term product stability.Non-limiting examples of lyoprotectants include sugars, such as sucroseor trehalose; an amino acid, such as monosodium glutamate,non-crystalline glycine or histidine; a methylamine, such as betaine; alyotropic salt, such as magnesium sulphate; a polyol, such as trihydricor higher sugar alcohols, e.g., glycerin, erythritol, glycerol,arabitol, xylitol, sorbitol, and mannitol; propylene glycol;polyethylene glycol; pluronics; and combinations thereof. The amount oflyoprotectant added to a pharmaceutical composition is generally anamount that does not lead to an unacceptable amount of degradation ofthe strain when the pharmaceutical composition is lyophilized.

In some embodiments, a bulking agent is included in the pharmaceuticalcomposition. The term “bulking agent” as used herein, includes agentsthat provide the structure of the freeze-dried product withoutinteracting directly with the pharmaceutical product. In addition toproviding a pharmaceutically elegant cake, bulking agents may alsoimpart useful qualities in regard to modifying the collapse temperature,providing freeze-thaw protection, and enhancing the strain stabilityover long-term storage. Non-limiting examples of bulking agents includemannitol, glycine, lactose, and sucrose. Bulking agents may becrystalline (such as glycine, mannitol, or sodium chloride) or amorphous(such as dextran, hydroxyethyl starch) and are generally used informulations in an amount from 0.5% to 10%.

Other pharmaceutically acceptable carriers, excipients, or stabilizers,such as those described in Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. (1980) may also be included in a pharmaceuticalcomposition described herein, provided that they do not adversely affectthe desired characteristics of the pharmaceutical composition. As usedherein, “pharmaceutically acceptable carrier” means any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed and include: additional bufferingagents; preservatives; co-solvents; antioxidants, including ascorbicacid and methionine; chelating agents such as EDTA; metal complexes(e.g., Zn-protein complexes); biodegradable polymers, such aspolyesters; salt-forming counterions, such as sodium, polyhydric sugaralcohols; amino acids, such as alanine, glycine, glutamine, asparagine,histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine,glutamic acid, and threonine; organic sugars or sugar alcohols, such aslactitol, stachyose, mannose, sorbose, xylose, ribose, ribitol,myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols(e.g., inositol), polyethylene glycol; sulfur containing reducingagents, such as urea, glutathione, thioctic acid, sodium thioglycolate,thioglycerol, [alpha]-monothioglycerol, and sodium thio sulphate; lowmolecular weight proteins, such as human serum albumin, bovine serumalbumin, gelatin, or other immunoglobulins; and hydrophilic polymers,such as polyvinylpyrrolidone.

The pharmaceutical composition may be prepared for oral, sublingual,buccal, intravenous, intramuscular, subcutaneous, intraperitoneal,conjunctival, rectal, transdermal, intrathecal, topical and/orinhalation-mediated administration. In a preferred embodiment, thepharmaceutical composition may be a solution which is suitable forintravenous, intramuscular, conjunctival, transdermal, intraperitonealand/or subcutaneous administration. In another embodiment, thepharmaceutical composition may be a solution which is suitable forsublingual, buccal and/or inhalation-mediated administration routes. Inan alternative embodiment, the pharmaceutical composition may be a gelor solution which is suitable for intrathecal administration. In analternative embodiment, the pharmaceutical composition may be an aerosolwhich is suitable for inhalation-mediated administration. In a preferredembodiment, the pharmaceutical composition may be prepared forintrathecal administration. The pharmaceutical composition may furthercomprise common excipients and carriers which are known in the state ofthe art. For solid pharmaceutical compositions, conventional nontoxicsolid carriers may be used which include, for example, pharmaceuticalgrades of mannitol, lactose, starch, magnesium stearate, sodiumsaccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, andthe like. For solution for injection, the pharmaceutical composition mayfurther comprise cryoprotectants, lyoprotectants, surfactants, bulkingagents, anti-oxidants, stabilizing agents and pharmaceuticallyacceptable carriers. For aerosol administration, the pharmaceuticalcompositions are generally supplied in finely divided form along with asurfactant and propellant. The surfactant must, of course, be nontoxic,and is generally soluble in the propellant. Representative of suchagents are the esters or partial esters of fatty acids containing from 6to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic,stearic, linoleic, linolenic, olesteric and oleic acids with analiphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, suchas mixed or natural glycerides may be employed. A carrier can also beincluded, as desired, as with, e.g., lecithin for intranasal delivery.For suppositories, traditional binders and carriers may include, forexample, polyalkalene glycols or triglycerides.

Medical Uses

In a seventh aspect, the present invention provides the AAV vector ofthe present invention, the nucleic acid of the present invention or thepharmaceutical composition of the present invention for use as amedicament. In an eight aspect, the present invention provides the AAVvector of the present invention, the nucleic acid of the presentinvention or the pharmaceutical composition of the present invention foruse in the treatment of Friedreich's ataxia.

In a preferred embodiment, the AAV vector of the present invention, thenucleic acid of the present invention or the pharmaceutical compositionof the present invention is administered intrathecally, intramuscularly,intracerebrally or intracerebroventricularly, preferably intrathecallyor intramuscularly.

In a preferred embodiment, the AAV vector of the present invention, thenucleic acid of the present invention or the pharmaceutical compositionof the present invention is administered at a dose of at least 1×10⁹vector genomes/Kg body weight, preferably 1×10¹⁰, 1×10¹¹ or 1×10¹²vector genomes/Kg body weight. More preferably, at least or about4.6×10¹² vector genomes/Kg body weight are administered.

EXAMPLES Example 1: Plasmid Construction

The coding sequence of the isoform 1 of human frataxin (hFXN) was fusedto a hemagglutinin tag (HA) and cloned into the pcDNA3.1 expressionvector using the with In-Fusion® HD Cloning Kit (Clontech). The fusionsystem was used for all the cloning steps. Several constructs weregenerated fusing either the CMV, or the human (h) promoters (p) ofsynapsin (phSYN), neuron-specific enolase (phNSE), 1,255 bp of the FXNpromoter (phFXN1255), or the human phosphoglycerate kinase isoform 1(phPGK1) with the coding region of the FXN gene. Further regulatoryelements such as the CMV enhancer and the Kozak sequences were added atthe 5 prime-end in addition to the woodchuck hepatitis virus responsiveelement (WPRE) sequence at the 3 prime-end. All these expression vectorsgenerated constructs are listed in Table 2.

Plasmids containing the same combination of regulatory elements werealso generated to drive luciferase expression by replacing the codingsequence of FXN for the firefly luciferase coding sequence (LUC) thatwas amplified from the pGL3-LUC vector (Promega) also using theIn-Fusion® HD Cloning Kit (Clontech). These plasmids are listed in Table3.

Example 2: Recombinant Adeno-Associated Viral Vector Construction andProduction

The expression cassettes from the pcDNA3.1-phPGK-kFXN-HA-WPRE and of thepcDNA3.1-phPGK-kLUC-HA-WPRE plasmid were cloned into the SnaBI-MfeIsites of the recombinant AAV9 vector (rAAV9-phPGK1-FXN-HA-WPRE vectorand rAAV9-phPGK-LUC-WPRE vector). In addition, a control null vector wasgenerated lacking the FXN coding sequence (AAV2/9-null). All threeconstructs and viral particles were generated by the Vector ProductionUnit at Center of Animal Biotechnology and Gene Therapy (UniversitatAutònoma de Barcelona). The final titers obtained were 1.4×10¹³ vg/mlfor AAV9-phPGK-FXN-HA-WPRE, 9.8×10¹² vg/ml for AAV9-phPGK-LUC-WPRE, and6×10¹² vg/ml for AAV2/9-null.

Example 3: Optimizing Frataxin Expression

1. Cell Culture, In Vitro Expression, and Luciferase Reporter Assay

Mouse neuroblastoma cells (N2a), Human neuroblastoma cells (SH-SY5Y) andHuman Embryonic Kidney (HEK 293) cells were cultured in Dulbecco'sModified Eagle's medium (DMEM) containing 10% Fetal bovine serum(Sigma), 2 mM glutamine, 50 μg/ml penicillin/streptomycin (Lifetechnologies) at 70% confluence in 10 cm culture dishes. Transfectionswere carried out using lipofectamine 2000 (Life technologies) for N2aand SH-SY5Y cells and calcium phosphate for HEK 293 using 4 μg plasmidDNAs alone in addition to 0.25 μg EGFP. Following the transfection, themedia was replaced with fresh DMEM culture media for the HEK cells andwith Neurobasal, B27 supplement, 10 μM retinoic acid, 2 mM glutamine, 50μg/ml Penicillin/Streptomycin for the N2a and SH-SY5Y cells. Forty-eighthours after the cell culture media change the cells transfected with theexpression plasmids listed in Table 2 were harvested and stored at −80°C. until further use. For luciferase reporter assay the plasmids listedin Table 3 were transfected as mentioned above and the cells werere-plated into 384 well plates and the luciferase activity determinedusing the dual luciferase reporter assay kit as per manufacturerinstructions (Promega).

2. SDS-PAGE and Immunoblotting

Proteins were extracted from cultured cells by homogenization in RIPAlysis buffer: 10 mM Tris-HCl pH 7.4, 140 mM NaCl, 0.1% sodiumdeoxycholate, 1% Triton X-100, 1% SDS, 2 mM Ethylenediaminetetraaceticacid (EDTA), 25 mM sodium fluoride (NaF), 2.5 mM NaVO3 and proteininhibitor cocktail (Roche). Protein concentration was determined usingthe DC-BioRad protein assay (BioRad). Total protein extract were mixedwith 4× Protein Sample Loading Buffer (Li-Cor Biosciences) containing 1mM DTT and separated by electrophoresis on a 15% acrylamide gel atconstant 20 mA before transfer to PVDF membranes. Primary antibodiesused were anti-FXN PAC 2518 (generous gift from by Dr. Grazia lsaya,Mayo Clinic, Rochester Minn.), anti-FXN (1G2, Merck Millipore), anti-HAtag (clone 16612, MMS-101P, Covance) and anti-beta actin (AC15, Sigma).Infrared-dye conjugated secondary antibodies were anti-mouse IRDye-800CWand anti-Rabbit IRDye 700CW (Li-Cor Biosciences) and theimmunoreactivity was detected using the Odyssey analyser software v2.1(Li-Cor Biosciences).

3. Results

As can be seen in FIGS. 1-5 and Tables 2-3, the provision of a constructwhich comprises the hPGK1 promoter, linker sequences (length andcontent) and the WPRE allowed for the expression of frataxin in cells.The human PGK1 promoter is a metabolically regulated promoter expressedin physiologically relevant tissues thereby making a good candidate as apromoter for frataxin. However the regulatory elements or the linkersequences/length to be used to be able to express the frataxin codingsequences was not clear. By trying different combinations of elementsand sequences our results show that the protein expression from thehuman frataxin coding sequences requires the inclusion of anoperationally functional linker between the hPGK1 promoter and the FXNcoding sequences since constructs containing a length of about 105 ntallow for FXN expression while linker lengths around 35 nt do not, evenwith the addition of the WPRE RNA stabilizer sequence. Interestingly,this linker requirement was only noted when using the hPGK1 promoter butnot with other promoters tested (ie. hSYN). Further, the levels offrataxin protein from this vector are much lower than that obtained fromthe CMV or CAG promoter driven vectors even in the absence of WPREsequences thereby being more comparable to endogenous levels. One of thelimitations of the previous approaches is that to be able to obtainfrataxin expression, high driving promoters such as CAG or CMV promotershave been used resulting in very high levels of protein expression whichmay cause cellular stress and override some of the protective effects.The vector presented here is able to drive the expression of frataxinprotein in relevant nervous system and peripheral tissues in quantitieswithin physiologically functional range.

TABLE 2 Summary of results. Relative levels of FXN after transfectionwith different constructs Frataxin expression plasmids Cell typeAntibody Immunoreactivity 1.1 pcDNA3.1-pCMV-FXN-HA N2A/HEK293/ Anti-HAtag Covance)/ ++ SHY Anti-FXN 1G2 (Merck Millipore) 1.2pcDNA3.1-phSYN-FXN-HA SHSY Anti-HA tag (Covance) − 1.3pcDNA3.1-phFXN-FXN-HA HEK293/SHSY Anti-HA tag (Covance) − 1.4pcDNA3.1-prNSE-FXN-HA N2A Anti-HA tag (Covance) − 1.5pcDNA3.1-phNSE-FXN-HA HEK293 Anti-HA tag (Covance) − Addition of WPRE2.1 pcDNA3.1-pCMV-FXN-HA-WPRE N2A/HEK293 Anti-HA tag (Covance)/ +++Anti-FXN 1G2 (Merck Millipore) 2.2 pcDNA3.1-phSYN-FXN-HA-WPRE N2A/HEK293Anti-HA tag (Covance) − 2.3 pcDNA3.1-phSYN-5′UTR_FXN- N2A/ HEK293Anti-HA tag | (Covance) − FXN-HA-WPRE 2.4 pcDNA3.1-phFXN-FXN-HA-WPREN2A/ HEK293 Anti-HA tag (Covance) − 2.5 pcDNA3.1-prNSE-FXN-HA-WPRE N2AAnti-HA tag (Covance) − Addition of CMV enhancer 3.1pcDNA3.1-E-phFXN-FXN-HA-WPRE HEK293 Anti-HA tag (Covance) − 3.2pcDNA3.1-E-phFXN-FXN-HA N2A/HEK293 Anti-HA tag (Covance) − 3.3pcDNA3.1-E-phSYN-FXN-HA-WPRE N2A Anti-HA tag (Covance) − Addition ofKOZAK and WPRE sequences 4.1 pcDNA3.1-pCMV-105nt-kFXN-HA-WPRE N2A/HEK293Anti-HA tag (Covance)/ ++++ Anti-FXN 1G2 (Merck Millipore) 4.2pcDNA3.1-phSYN.kFXN-HA-WPRE HEK293/N2A Anti-HA tag (Covance) −/+ 4.3pcDNA3.1-prNSE-kFXN-HA-WPRE HEK293/N2A Anti-HA tag (Covance) −/+ 4.4pcDNA3.1-phFXN-kFXN-HA-WPRE HEK293 Anti-HA tag (Covance) − 4.5pcDNA3.1-E-phFXN-kFXN-HA-WPRE HEK293 Anti-HA tag Covance) − Use ofspecific linker, KOSAK and WPRE sequences 5.1pcDNA3.1-phPGK-105nt-kFXN-HA-WPRE N2a/HEK293 Anti-HA tag| (Covance)/ ++Anti-FXN 1G2 (Merck Millipore) 5.2 pcDNA3.1-phNSE-105nt-kFXN-HA-WPREN2a/HEK293 And-HA tag (Covance) + 5.3 pcDNA3.1-phSYN-105nt-kFXN-HA-WPREN2a/HEK293 Anti-HA tag (Covance) − 5.4 pcDNA3.1-phFXN-105nt-kFXN-HA-WPREN2A/HEK293 Anti-HA tag (Covance) −

TABLE 3 Reporter assay of frataxin and PGK1 promoter constructsLuciferase expression plasmids Cell type ExpressionpcDNA3.1-pCMV-135-nt-LLIC-WPRE . N2a/HEK293 ++++ pcDNA3.1-phPGK-135nt-LUC -WP RE N2a/HEK293 ++ pcDNA3.1-E-phPGK-135nt-LUC-WPRE N2a++ pcDNA3.1-phFXN1255-135nt-LUC-WPRE HEK293 −pcDNA3.1-phFXN1255-35nt-LUC-WPRE N2a/HEK293 −pcDNA3.1-phFXN220-35nt-LUC-WPRE N2a/HEK293 +pcDNA3.1-phEXN1255OCT-35nt-LUC-WPRE N2a/HEK293 −

Example 4: In Vivo Data

1. Animals

The Friedreich Ataxia mouse model YG8R developed by Pook and colleagues(Pook et al. 2001. Neurogenetics. 3(4):185-93; Virmouni et al., 2014.PLoS ONE. 9(9):e107416) used in this study was obtained from the JacksonLaboratories Repository (Stock no. 012253). This human FXN transgenicmouse model is a knockout for the endogenous mouse frataxin gene(Fxn−/−) and contains the human FXN YAC transgene from a founder YG8(carrying two tandem copies of the human FXN gene with approximately 82and with 190 GAA trinucleotide sequence repeats). Mice were housed withSPF-Like conditions in NexGen Mouse IVC cages (Allentown, N.J.) with a12-h light-dark cycle and controlled negative pressure, temperature andhumidity, and free access to water and an irradiated rodent chow Tekladglobal 18% protein (Envigo). The YG8R mouse colony was generated at TheInstitute for Health Science Research Germans Trias i Pujol (IGTP).Female and male hemizygous YG8R (Tg/−) and the WT C57Bl/6 mice were usedin all experiments.

Mice hemizygous for the mutant human FXN gene (YG8R) were genotyped aspreviously reported (29,36). Briefly, the genomic DNA was extracted fromthe YG8R mouse tail by Maxwell® 16 Mouse Tail DNA Purification Kit(Promega). The transgene copy number was determined for each mouse usingquantitative real-time PCR (qPCR) in LightCycler® 480 Instrument (Roche)using SYBR Premix Ex Taq II (Tli RNase H Plus) (Takara) and thefollowing primers for human frataxin transgene: hFXN_Tg_FW:5′-GAAC-TTCAAATTAGTTCCCCTTTCTTC-3′ (SEQ ID NO: 7), hFXN_Tg_RV:5′-CACAGCCAT-TCTTTGGGTTTC-3′ (SEQ ID NO: 8); and internal controlApolipoprotein B-100 isoform X1: IC_FW: 5′-CACGTGGGCTCCAGCATT-3′ (SEQ IDNO: 9), IC_RV: TCACCAGTCATTTCTGCCTTTG (SEQ ID NO: 10). The assay wasperformed with thermal cycling conditions: 95° C. for 5 minutes, and 40cycles of 95° C. for 20 seconds and 60° C. for 30 seconds, 72° C. for 30seconds and finally 1 cycle at 72° C. 2 min. Samples were assayed intriplicate for each gene of interest and levels of the transgenedetermined by the Ct (ΔΔCt) method. The transgene copy numbers wasestimated relative to the Ct data from the control sample with a knowncopy number. The GAA repeat length was determined by GAA PCRamplification PCR using LA taq (Invitrogen) and the following primers:GAA-F: 5′-GGGATTGGTT-GCCAGTGCTT-AAAAGTTAG-3′ (SEQ ID NO: 11) and GAA-R:5′-GATCTAAGGACCATCATGG-CCACACTTGCC-3′ (SEQ ID NO: 12). PCR products wereresolved in 1.5% agarose gels by electrophoresis at 100 V for 3 hoursand the band sizes were analysed. The number of GAA repeats were thendetermined by subtracting 451 bp (flanking non-repeat DNA) from the PCRproduct size, and dividing the remaining base pair repeat size by 3.

All animal procedures were carried out in accordance with EU and localregulations and approved by the appropriate local Ethics Committees.

2. AAV Administration

YG8R hemizygous and WT 10-weeks-old mice were anesthetized byintraperitoneal injection of ketamine (10 mg/kg of body weight; Imalgene500; Rhône-Merieux, Lyon, France) and xylazine (1 mg/kg of body weight;Rompun; Bayer). Intrathecal administration of theAAV9-phPGK-FXN-HA-WPRE, AAV9-phPGK-LUC-WPRE, or AAV2/9-null wasperformed at the lumbar region. After lateral spine exposure, byparavertebral muscle dissection, viral vectors were slowly injected intothe CSF through a 33-gauge needle and a Hamilton syringe between lumbarvertebrae L3 and L4. The appropriate access to the intrathecal space wasconfirmed by the animal's tail movement. Thereafter the muscle and skinwere sutured. The quantity administered for each mouse of the differentviral vectors was 4.6×10e-12 vg/Kg mouse weight.

3. Vector Bio-Distribution Using Luciferase Imaging

For in vivo evaluation, of the AAV9-phPGK-LUC-WPRE vectorbiodistribution, the mice were administered intrathecally with thevector at 10 weeks of age (2.5 months-old) and 3.5 months later theyreceived a single intraperitoneal injection of D-luciferin substratesolution at 150 mg/kg of mouse weight. The mice were anesthetised 15 minafter substrate administration by inhaled anaesthesia (isoflurane 4% forinduction and 2% for maintenance). Anesthetized mice were maintained inthe dark chamber of Perkin Elmer Ivis Lumina II (Caliper Life Sciences,Germany) to record the photon emissions. Images were analysed with theLiving imaging software (Xenogen Corporation, CA, EUA) with 1 minintegration time, 12.5 cm vision field. For ex-vivo evaluation, tissuesand organs of mice were extracted after 15 min of substrateadministration. Tissues and organs were placed into clear dishes andimages were captured using the Living imaging software (XenogenCorporation, CA, EUA) as above. Results can be seen in FIG. 6.

4. qRT-PCR

RNA was extracted from 30 mg of freshly frozen mouse tissue using theRNeasy Mini Kit (Qiagen). The RNA RIN and quantification was obtainedwith an Agilent 2200 TapeStation (Agilent). RNA was retrotranscribed tocDNA (25 ng/ul) using PrimeScript™ RT reagent Kit (Takara) with thermalconditions: 37° C. for 15 minutes, 85° C. for 5 seconds. The qRT-PCR wasperformed with cDNA from mouse tissues to test levels of frataxinexpression in a multiwell plate format using the LightCycler480instrument (Roche Diagnostics). Reaction mixtures contained a totalvolume of 10 μl consisting of 0.1 mM of each primer, 10 ng of cDNA and 5μl of TaqMan Universal Master Mix II, no UNG (Thermofisher Scientific).Primer-probe for human frataxin detection were predesigned by Bio-rad(dHsaCPE5031641, Bio-rad), primers for Beta-2 microglobulin (62m)housekeeping gene for mice samples were predesigned by IDT(Mm.PT.39a.22214835; IDT). The assay was performed with thermal cyclingconditions: 50° C. for 2 minutes, 95° C. for 10 minutes, and 40 cyclesof 95° C. for 15 seconds and 60° C. for 1 minute. Samples were assayedin triplicate for each gene of interest and the levels determined by theCt (ΔΔCt) method. Results can be seen in FIG. 7.

5. SDS-PAGE and Immunoblotting

Proteins were extracted from mouse tissue by homogenization in RIPAlysis buffer: 10 mM Tris-HCl pH 7.4, 140 mM NaCl, 0.1% sodiumdeoxycholate, 1% Triton X-100, 1% SDS, 2 mM Ethylenediaminetetraaceticacid (EDTA), 25 mM sodium fluoride (NaF), 2.5 mM NaVO3 and proteininhibitor cocktail (Roche). Protein concentration was determined usingthe DC-BioRad protein assay (BioRad). Total protein extract were mixedwith 4× Protein Sample Loading Buffer (Li-Cor Biosciences) containing 1mM DTT and separated by electrophoresis on a 15% acrylamide gel atconstant 20 mA before transfer to PVDF membranes. Primary antibodiesused were anti-FXN PAC 2518 (generous gift from by Dr. Grazia lsaya,Mayo Clinic, Rochester Minn.), anti-FXN (1G2, Merck Millipore), anti-HAtag (clone 16612, MMS-101P, Covance) and anti-beta actin (AC15, Sigma).Infrared-dye conjugated secondary antibodies were anti-mouse IRDye-800CWand anti-Rabbit IRDye 700CW (Li-Cor Biosciences) and theimmunoreactivity was detected using the Odyssey analyser software v2.1(Li-Cor Biosciences). Results can be seen in FIG. 8.

6. Clasping

Hindlimb clasping has been shown to be a marker of disease progressionin a number of mouse models of neurodegeneration. Each mouse was liftedby the tail away from any surrounding objects. The hindlimb position wasobserved for 10 seconds and scored as follows: If the hindlimbs wereconsistently splayed outward, away from the abdomen, it is assigned ascore of 0. If one hindlimb was retracted toward the abdomen for morethan 50% of the time suspended, it receives a score of 1. If bothhindlimbs were partially retracted toward the abdomen for more than 50%of the time suspended, it receives a score of 2. If its hindlimbs wereentirely retracted and touching the abdomen for more than 50% of thetime suspended, it receives a score of 3. The clasping reflex wasassessed every 2 months in mice from 4 months of age. Results can beseen in FIG. 9.

7. Electrophysiology

Amplitude (μV) and nerve conduction velocity (m/s) were measured in thecaudal nerve of the mouse's tail. With animals under inhaled anaesthesia(isoflurane 2%), the stimulation electrode needle was situatedsubcutaneously at 4 different points of stimulation (1 cm, 2 cm, 3 cm, 4cm from tail tip) while the registration needle point was fixed at 6 cmfrom tail tip. Values were recorded with the EMG/PE N-EP with 2 channelsfrom Medelec Synergy apparatus (Viasys, EUA). Duringelectrophysiological tests, the skin temperature of the animals wasmaintained above 32° C. The mice were assessed every 2 months from 4months of age. Results can be seen in FIG. 10.

8. Results

We propose that the presented AAV treatment would be effective astreatment for FRDA because it can be expressed in physiologicallyrelevant tissues, levels, shows similar processing as the endogenousprotein and can significantly improve the electrophysiologicalproperties of affected neurons and neurological symptoms in a mousemodel of FRDA (YG8R). FIG. 6 shows that the luciferase gene in the AAV9vector under the same regulatory elements than the FXN in theAAV9-hPGK1-FXN vector when injected intrathecally, is expressedthroughout the spinal cord, brain as well as in peripheral tissues. Alsoexpression of a luciferase under the same vector and regulatory elementsstay consistent around at least 7 months after injection (data noshown). Because diminished levels of frataxin underlie most if not allthe symptoms in FRDA and individuals carriers for the mutationexpressing ≥25% levels of frataxin are free of FRDA symptoms, a smallincrease in the FXN expression should be able to prevent orsignificantly ameliorate symptoms in FRDA patients. However, very highlevels of FXN expression or any other protein could induce cellular andeventually diminish the protective effects or even worsen the symptoms.Therefore, it is of interest to develop a FXN expressing vector thatresults in levels as similar as possible to those of the endogenous FXNprotein. Therefore we developed a vector (AAV9-hPGK1-FXN) vector thatallows for the expression of FXN under a metabolically regulatedpromoted in physiologically relevant levels and delivered itintrathecally into 10-week-old YG8R hemizygous mice (Tg/−). Higherlevels of FXN mRNA were detected in the YG8R hemizygous mice (Tg/−) 3.5months after intrathecal injection of the AAV9-PGK1-FXN vector comparedto non-injected mice both in the liver and thoracic portion of thespinal cord (0.5 and 2-fold, respectively; FIG. 7). These were equal orlower than in the homozygous YG8R mice which showed 0.5 and 3-foldspinal cord, respectively compared to the hemizygous YG8R mice. Thisindicates that this FXN encoding vector injected in the L3-L4intrathecal region was taken-up and expressed in thoracic spinal cordneurons by either retrograde transport or as it diffuses through thespinal fluid. We also noted that the AAV9 vector intrathecallyadministered is also able to cross the blood brain barrier and reachperipheral tissues such as liver, heart, etc. (FIG. 6) and is able toexpress the hFXN mRNA from the injected vector (FIG. 7).

The levels of FXN protein expression from the recombinant vectordeveloped was also analyzed. Although the antibody used shows a higheraffinity for the human FXN protein as seen by the protein patternobtained after 1 hour versus overnight incubation of the blot with theprimary anti-FXN antibody, FIG. 8 shows that the levels of the FXNprotein expressed from the rAAV9-PGK1-FXN vector appear similar to thoseof the mouse FXN levels in the WT mice (FIG. 8A, lanes 3 vs. 4 and FIG.8B second panel, lanes 3 vs. 4 and 5 vs. 6 and 7). Remarkably, we wereable to detect the recombinant FXN protein in the motor cortex region(C1) of the mouse brain (FIG. 8B, lane 7). This indicates that thedelivery of our vector by intrathecal injection into the lumbar regionof the spinal cord results in the distribution of the vector throughoutmotor related regions of the CNS. In addition, the recombinant proteinappears to be processed in a similar pattern as the wild-type FXNprotein with the intermediate and mature form being the most prominentprocessed form.

We further analysed the phenotype of the YG8R hemizygous mice (Tg/−)treated with the rAAV9-PGK1-FXN compared to the rAAV9-null vector forthe clasping neurological reflex and the electrophysiological propertiesof the caudal nerve. The clasping reflex has been shown to be involvedin several neurodegenerative disorders. This reflex appears to involvesensory and also spinal motor pathways regulating the fore and hindlimbmovements. In particular, the cerebello-cortico-reticular pathways havebeen shown to be involved. Therefore, to determine the effect of thetreatment of the rAAV9-hPGK1-FXN vector on these pathways in the YG8Rmice we quantified the clasping reflex at different times following theintrathecal injection of the vector (see methods). FIG. 9 shows that theYG8R mice treated with either the AAV9-null and AAV9-FXN vectors exhibitclasping defects as early as four-months of age (1.5 months afterintrathecal injection). However, overtime the clasping reflex isnormalized in the AAV-FXN treated mice to more closely resemble those ofthe WT mice even more so at 10-months of age (6.5 months afterinjection). This suggests that there are early anomalies in the sensoryand cerebello-cortico-reticular system in the FRDA mouse model that aresignificantly ameliorated or reversed by the treatment with the AAV-FXNvector.

To more specifically determine the effects of the treatment on thesensory neurons we determined the electrophysiological properties(amplitude and velocity) of the caudal nerve of the YG8R mice treatedwith the AAV-FXN vector compared with those treated with a AAV9-nullcontrol vector. FIG. 10 shows that at all distances from the tail tip(1-4 cm) no significant differences were noted between the null vectortreated WT or the null and AAV-FXN treated YG8R hemizygous mice at fourmonths of age (1.5 months following treatment). However, from 6 monthsof age on, while the AAV9-null treated YG8R (Tg/−) mice showedsignificant decline in amplitude, the AAV9-FXN treated mice were moreclosely resembling the WT mice even as late as 13 months of age (9.5months after treatment). No changes in velocity were noted (data notshown). These data indicate that the treatment with the AAV-hPGK1-FXNvector was able to preserve the function of the caudal nerve in the YG8Rmice possibly by preventing loss of the axons from the affected neurons.

1. An adeno-associated virus (AAV) vector comprising a nucleic acid,wherein the nucleic acid comprises: (i) a nucleic acid sequence encodingfrataxin; (ii) a phospho-glycerate-kinase (PGK) promoter consisting ofSEQ ID NO:1; and (iii) a woodchuck hepatitis virus posttranscriptionalregulatory element (WPRE); wherein (ii) and (iii) are operably linked toand regulate the expression of (i), wherein there is an operationallyfunctional linker between (i) and (ii), wherein said linker consists ofSEQ ID NO: 6, and wherein the AAV vector is an AAV serotype 9 vector. 2.The vector according to claim 1, wherein the nucleic acid sequenceencoding frataxin comprises SEQ ID NO:3, or a sequence which is at least90% identical to SEQ ID NO:3 and is a functional variant of frataxin. 3.The vector according to claim 1, wherein the nucleic acid sequenceencoding frataxin consists of SEQ ID NO:3, or a sequence which is atleast 98% identical to SEQ ID NO:3 and is a functional variant offrataxin.
 4. The vector according to claim 1, wherein the WPRE consistsof SEQ ID NO: 2 or a sequence which is at least 90% identical to SEQ IDNO:2.
 5. The vector according to claim 1, wherein the WPRE consists ofSEQ ID NO: 2 or a sequence which is at least 99% identical to SEQ ID NO:2.
 6. The vector according to claim 1, wherein the nucleic acid sequenceencoding frataxin consists of SEQ ID NO:3, and the WPRE consists of SEQID NO: 2 or a sequence which is at least 90% identical to SEQ ID NO: 2.7. The vector according to claim 1, wherein the nucleic acid sequenceencoding frataxin consists of SEQ ID NO:3, and the WPRE consists of SEQID NO: 2 or a sequence which is at least 99% identical to SEQ ID NO: 2.8. The vector according to claim 1, wherein the sequence of the nucleicacid which comprises (i), (ii), (iii) and the linker, consists of SEQ IDNO: 4 and, wherein the AAV vector is an AAV serotype 9 vector.
 9. Atransfer vector which comprises a nucleic acid comprising: (i) a nucleicacid sequence encoding frataxin; (ii) a PGK promoter consisting of SEQID NO:1; and (iii) a WPRE; wherein (ii) and (iii) are operably linked toand regulate the expression of (i), wherein there is an operationallyfunctional linker between (i) and (ii), and wherein said linker consistsof SEQ ID NO: 6, and wherein the transfer vector further comprisesadditional nucleic acid elements for promoting integration ortransposition of the transfer vector into AAV serotype 9 vector. 10-12.(canceled)
 13. A pharmaceutical composition comprising the AAV vectoraccording to claim 1 and a pharmaceutically acceptable carrier ordiluent.
 14. (canceled)
 15. A method of treating Friedrich's ataxia, themethod comprising administering a therapeutically effective amount ofthe AAV vector according to claim 1 to a subject in need thereof.